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Clinical and Experimental Immunology Jul 2018Studies of cerebrospinal fluid (CSF) γδ T cells in children are limited, due especially to the lack of control data. In adults, gamma/delta T cells (TCR-γδ)...
Studies of cerebrospinal fluid (CSF) γδ T cells in children are limited, due especially to the lack of control data. In adults, gamma/delta T cells (TCR-γδ) residing in the intrathecal space are sometimes involved in neuroinflammation. To evaluate the possible role of γδ T cells in paediatric neuroinflammation, we immunophenotyped cerebrospinal fluid (CSF) and blood lymphocytes using flow cytometry in a case-control study of 100 children with non-inflammatory neurological disorders (NIND), 312 with opsoclonus-myoclonus (OMS) and 23 with other inflammatory neurological disorders (OIND). In NIND, the negative correlation between CSF γδ T cell frequency and patient age was striking: median frequency of 27% in infants and 3·3% in teens. Interindividual variations were largest in the youngest. There was no gender effect. In all OMS, after correcting for age, only a small effect of OMS severity remained. Measurement of markers for γδ T cell activation [human leucocyte antigen D-related (HLA-DR)], maturation (CD45RA, CD45RO) or intracellular cytokine staining [interleukin (IL)-4, interferon (IFN)-γ] failed to discriminate OMS and NIND groups. Of seven OMS immunotherapies/combinations, none altered the frequency of total CSF γδ T cells or subsets significantly. In OIND, the CSF γδ T cell frequency was < 10% for single samples of other paraneoplastic disorders [anti-neuronal nuclear antibody (ANNA)-1, PCA-1, teratoma-associated syndrome], cerebellar ataxia (post-infectious, ataxia-telangiectasia), acute disseminated encephalomyelitis, neuroborreliosis and encephalitis. This study provides new insights into CSF γδ T cells in the paediatric population. Although their role in CSF remains elusive, the negative age correlation, resistance to immunotherapy and our age cut-off references for NIND are important findings for the design of future paediatric studies.
Topics: Age Factors; Case-Control Studies; Cerebrospinal Fluid; Child; Child, Preschool; Female; Flow Cytometry; HLA-DR Antigens; Humans; Infant; Inflammation; Interferon-gamma; Interleukin-4; Leukocyte Common Antigens; Lymphocyte Activation; Male; Nervous System Diseases; Opsoclonus-Myoclonus Syndrome; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes
PubMed: 29485697
DOI: 10.1111/cei.13122 -
Viruses Jan 2018Zika virus (ZIKV) is an emerging pathogen from the family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including...
Zika virus (ZIKV) is an emerging pathogen from the family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.
Topics: Animals; Brazil; Canada; Cell Line; Cell Survival; Chlorocebus aethiops; Communicable Diseases, Imported; Cytopathogenic Effect, Viral; Genome, Viral; Humans; Mutation; Polyproteins; RNA, Viral; Thailand; Vero Cells; Viral Load; Zika Virus; Zika Virus Infection
PubMed: 29382068
DOI: 10.3390/v10020053 -
Scientific Reports Oct 2017Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been...
Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4CD25 Treg cells and other CD4CD25 regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4 T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.
Topics: Animals; Antibodies, Monoclonal; Apoptosis; B7-H1 Antigen; CD4 Antigens; Cell Proliferation; Cytotoxicity, Immunologic; Female; Immunologic Factors; Immunotherapy; Interleukins; Lymphocyte Depletion; Mice; Mice, Inbred A; Neuroblastoma; Programmed Cell Death 1 Receptor; T-Lymphocytes, Regulatory; Tumor Cells, Cultured
PubMed: 29070883
DOI: 10.1038/s41598-017-14417-6 -
Cancer Cell Oct 2017Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology...
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.
Topics: Adolescent; Adult; Animals; Cell Adhesion; Chick Embryo; Child; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Staging; Nerve Tissue Proteins; Neuroblastoma; Receptors, Cell Surface; Semaphorins; Tumor Microenvironment
PubMed: 29017055
DOI: 10.1016/j.ccell.2017.09.006 -
International Journal of Cancer Jan 2018Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs)...
Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and relapse. Although these cells represent a major obstacle in the treatment of neuroblastoma patients, insights into their expression profile remained elusive. The present RNA-Seq study of stage 4/M primary tumors, enriched BM-derived diagnostic and relapse DTCs, as well as the corresponding BM-derived mononuclear cells (MNCs) from 53 patients revealed 322 differentially expressed genes in DTCs as compared to the tumors (q < 0.001, |log FC|>2). Particularly, the levels of transcripts encoded by mitochondrial DNA were elevated in DTCs, whereas, for example, genes involved in angiogenesis were downregulated. Furthermore, 224 genes were highly expressed in DTCs and only slightly, if at all, in MNCs (q < 8 × 10 log FC > 6). Interestingly, we found the transcriptome of relapse DTCs largely resembling those of diagnostic DTCs with only 113 differentially expressed genes under relaxed cut-offs (q < 0.01, |log FC|>0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4 and GLTSCR2. This first RNA-Seq analysis of neuroblastoma DTCs revealed their unique expression profile in comparison to the tumors and MNCs, and less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected downregulation of genes encoded by chromosome 19. As these alterations might be associated with treatment failure and disease relapse, further functional studies on DTCs should be considered.
Topics: Biomarkers, Tumor; Bone Marrow Neoplasms; Disease Progression; High-Throughput Nucleotide Sequencing; Humans; Neoplastic Cells, Circulating; Neuroblastoma; Prognosis; Transcriptome
PubMed: 28921546
DOI: 10.1002/ijc.31053 -
Cancer Cell Sep 2017A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an...
A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.
Topics: Animals; Animals, Genetically Modified; Carcinogenesis; Cell Line, Tumor; Cell Movement; DNA-Binding Proteins; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; Hyperplasia; LIM Domain Proteins; Models, Biological; N-Myc Proto-Oncogene Protein; Neoplasm Invasiveness; Neoplasm Metastasis; Neuroblastoma; Signal Transduction; Transcription Factors; Transgenes; Zebrafish
PubMed: 28867147
DOI: 10.1016/j.ccell.2017.08.002 -
Proceedings of the National Academy of... Aug 2017Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue...
Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.
Topics: Animals; Antineoplastic Agents, Immunological; Cell Line, Tumor; Female; Gene Silencing; Glypicans; HEK293 Cells; Humans; Mice; Mice, Nude; N-Myc Proto-Oncogene Protein; Neuroblastoma; Single-Chain Antibodies; Wnt Signaling Pathway; Wnt3A Protein; Xenograft Model Antitumor Assays
PubMed: 28739923
DOI: 10.1073/pnas.1706055114 -
Oncology Letters Jun 2017Disseminated carcinomatosis of the bone marrow (DCBM) is a condition in which bone marrow (BM) metastases diffusely invade the BM, and is frequently accompanied by...
Disseminated carcinomatosis of the bone marrow with disseminated intravascular coagulation as the first symptom of recurrent rectal cancer successfully treated with chemotherapy: A case report and review of the literature.
Disseminated carcinomatosis of the bone marrow (DCBM) is a condition in which bone marrow (BM) metastases diffusely invade the BM, and is frequently accompanied by disseminated intravascular coagulation (DIC). While prostate, lung, breast and stomach malignancies, in addition to neuroblastoma, are the most prevalent non-hematological malignancies to metastasize frequently to the BM, colorectal cancer is a malignancy that rarely metastasizes to the BM. The present case describes a 65-year-old male patient treated by resection and one course adjuvant chemotherapy for stage IIIC rectal cancer who presented with nasal bleeding at 8 months post-surgery. A blood test exhibited DIC. A BM biopsy was performed and the definitive diagnosis was DCBM with DIC. Promptly, anti-DIC treatment and chemotherapy with a modified FOLFOX6 (folinic acid, leucovorin (LV), 5-fluorouracil (5-FU) and oxaplatin) regimen was started. Following 1 cycle of chemotherapy, DIC was improved and subsequent to 2 cycles of modified FOLFOX6 the patient was discharged. The patient was alive 263 days subsequent to the diagnosis of DIC, but succumbed to carcinomatous meningitis as a result of disease progression. To the best of our knowledge, this is the first report of DCBM with DIC of curatively resected rectal cancer as the first presentation of relapse that was successfully treated with aggressive therapy, including chemotherapy.
PubMed: 28599429
DOI: 10.3892/ol.2017.5983 -
Journal of Virological Methods Sep 2017Maintaining a healthy, continuous immortalized cell line is essential for rabies laboratories that perform virus isolation assays and test for the presence of viral...
Maintaining a healthy, continuous immortalized cell line is essential for rabies laboratories that perform virus isolation assays and test for the presence of viral neutralizing antibodies. Individuals who routinely work with rabies virus, such as rabies laboratory employees, or those who may have a high potential for exposure to rabies virus, including veterinarians, should be tested for the presence of anti-rabies viral neutralizing antibodies (VNA) every 6-24 months, depending on potential exposure level. The gold standard for serum neutralization assays require the use of live rabies virus and cells that are sensitive to rabies virus infection. Additionally, virus isolation assays are routinely performed in rabies laboratories as a back-up for the direct fluorescent antibody test (dFAT). Currently there are no guidelines or publications recommending the use of low, intermediate, or high passage cell lines in rabies assays. In this study, we compared the sensitivity of intermediate, high, and extremely high passaged neuroblastomas to rabies virus using virus isolation, serum neutralization, and real time RT-PCR techniques. Additionally, cells were examined microscopically to determine changes in morphology and dissemination of rabies virus antigen between intermediate, high, and extremely high passage cells. No significant difference was found between cell passage numbers and viral susceptibility between intermediate and high passaged cells. However, extremely high passaged cells (≥1200 passages) were less susceptible to viral infection and/or produced less virus following inoculation. As a result, rabies laboratories that use viral isolation and serum neutralization assays should regularly assess cell susceptibility to ensure the integrity and repeatability of the test.
Topics: Cell Line, Tumor; Humans; Neurons; Neutralization Tests; Rabies virus; Serial Passage; Tissue Culture Techniques; Viral Tropism; Virus Cultivation
PubMed: 28506631
DOI: 10.1016/j.jviromet.2017.05.005 -
Oncotarget Jun 2017Neuroblastoma (NB), ganglioneuroblastoma intermixed (GNBi) and ganglioneuroblastoma nodular (GNBn) are neuroblastic tumors that present with a wide range of symptoms and...
Neuroblastoma (NB), ganglioneuroblastoma intermixed (GNBi) and ganglioneuroblastoma nodular (GNBn) are neuroblastic tumors that present with a wide range of symptoms and variable prognoses. We retrospectively reviewed the pretreatment clinical (age, sex and tumor stage) and biological (MYCN amplification; and levels of lactate dehydrogenase, ferritin and neuron-specific enolase) characteristics of 279 patients who were diagnosed with pathologically confirmed NB and GNB from January 2005 to December 2015. The median age at diagnosis increased with grade of differentiation (NB: 28.9 months; GNBn: 38.4 months; GNBi: 47.5 months; p < 0.01). NB patients were more frequently diagnosed with adrenal tumors and had a higher prevalence of abnormal serum ferritin at the time of diagnosis (60.0% vs. 40.0% vs. 12.0%, P<0.001), NSE (96.0% vs. 93.0% vs. 81.0%, P=0.013) when compared with GNBn and GNBi patients. The prevalence rates of disseminated tumors and MYCN amplified tumors were lower in the GNBi group than in the GNBn and NB groups (13.0% vs. 25.0% vs. 44.0%, P=0.002; 0 vs. 14.0% vs. 26.0%, P=0.032, respectively). The overall survival (OS) of patients with GNB was significantly better than that of patients with NB (GNBi: 100%, GNBn: 74.5±11.4%, NB: 50.8±4.5%, respectively; P<0.01). Our study revealed that both NB and GNB have a wide range of presentations, and clinicians should be aware of both typical and atypical symptoms and signs. Children with GNB (especially GNBi) were more likely to present favorable prognostic factors than their NB counterparts, which consequently lead to better outcomes and longer survival for these patients.
Topics: Adult; Female; Ganglioneuroblastoma; Humans; Male; Neuroblastoma; Retrospective Studies; Survival Analysis
PubMed: 28465480
DOI: 10.18632/oncotarget.17146